The effect of fiber alignment and heparin coating on cell infiltration into nanofibrous PLLA scaffolds
Introduction
Dermal wound healing is a complex process requiring coordination of several biological processes, including ingrowth of cells, organization of extracellular matrix (ECM), regulation of inflammation, and rapid wound coverage to prevent infection [1]. For many wounds however, the “repair” mechanism often dominates over a more desirable “regeneration” mechanism, resulting in the formation of disorganized scar tissue instead of well-ordered skin [2]. One potential solution to this problem is to create tissue engineered scaffolds with properties that can enhance the natural wound healing process, such as aligned, organized, or bioactive fibrils to replace lost/damaged ECM and to guide new cells directly into the wound area with improved speed and overall organization.
Native ECM contains biological fibrils with diameters ranging from tens of nanometers to micrometers in scale. The organized structure of these matrix fibrils guides tissue morphogenesis and remodeling. In addition, matrix fibrils serve as “depots” for the storage of bioactive factors for the regulation of cell migration, proliferation and differentiation. Synthetic polymers can be specifically engineered (both physically and chemically) to aid tissue regeneration, and it is known that the surface microstructure and chemistry of these engineered substrates can influence the ability of the cells and tissues to attach, grow and function [3]. Electrospinning technology has been used to fabricate nonwoven nanofibrous scaffolds from biological and/or synthetic polymers, and has tremendous potential for tissue engineering applications [4], [5], [6], [7]. The diameter of the individual fibers can be specifically controlled down to the nanometer range, and the fibers can be patterned through a variety of methods [8], [9], [10]. Moreover, the electrospun nanofibers have large surface area to volume ratio, which allows for the direct attachment of ECM ligands, growth factors and other biomolecules onto fiber surfaces to locally modulate cell and tissue function and to guide and enhance regeneration. Although the patterning of the nanofibers has been shown to influence the alignment of cells and cellular processes [11], [12], [13], [14], [15], the effects of patterned and bioactive nanofibers on cellular migration and dermal wound healing have not been fully elucidated, especially with regard to the infiltration of cells into three-dimensional (3-D) scaffolds.
One limitation of electrospun nanofibrous scaffolds is the relatively small pore size (in comparison to the average diameter of most cells) and the resultant difficulty for cell infiltration into the 3-D structure, which retards matrix remodeling and tissue regeneration. Recently, salt-leaching methods have been used to increase the pore size of the electrospun scaffolds [16], but these methods still face the problems of collapse of fibrous structure after removing the salt. Our previous study showed excellent cell infiltration into vascular grafts with nanofibers aligned in the circumferential direction [11], but whether aligned fibers enhanced cell infiltration was not clear. In this study, we fabricated aligned nanofibrous scaffolds and studied their effect on cell infiltration in vitro and in vivo by using a dermal wound healing model. In addition, chemical modification of nanofibers could affect cell infiltration by directing cellular behavior and modifying physiological conditions. Since heparin can bind to many growth factors (e.g., basic fibroblast growth factor, epidermal growth factor, etc.) and matrix proteins (fibronectin) and can prevent clotting, we also investigated whether heparin modification of nanofibers can enhance dermal cell infiltration into 3-D scaffolds.
Section snippets
Electrospinning of nanofibrous polymer scaffolds
We used biodegradable poly(l-lactide) (PLLA) (1.09 dL/g inherent viscosity) (Lactel Absorbable Polymers, Pelham, AL) to fabricate nanofibrous scaffolds by electrospinning, as described previously [11], [12], [13]. The PLLA (10% w/v) solution (dissolved in hexafluoroisopropanol (HFIP) solvent) was delivered by a programmable pump to an electrically charged needle, which formed a nanoscale polymer fiber at the needle tip. The electrostatically charged fiber was ejected toward a grounded collecting
Electrospinning of nanofibrous scaffolds and heparin modification
Before using the nanofibrous membranes for biological experimentation, they were characterized for desired fiber architecture and chemical functionalization. As shown in Fig. 1A, B, we were able to obtain nanofibrous scaffolds with either random or aligned nanofibers by adjusting the rotation speed of the drum in electrospinning system, as described. Furthermore, toluidine blue staining verified the immobilization of heparin on the nanofibers (Fig. 1C).
Effects of nanofiber alignment and heparin modification on cell infiltration in vitro
To investigate the effects of nanofibrous
Discussion
Nanofibrous materials created by electrospinning have enormous potential for tissue engineering since they can mimic the structure and function of native ECM. These nanoscale fibers are similar in dimension to natural collagen fibers found throughout the ECM, and can be fabricated with varying alignment and orientation. In terms of wound healing, nanofibers have been characterized both in vitro [18], [19], [20] and in preliminary studies with rats [21] and guinea pigs [22], but these were
Conclusions
Aligned nanofibers can enhance cell infiltration into 3-D scaffolds in vitro and in vivo. Heparin modification of nanofibers promotes cell infiltration into 3-D scaffolds in vivo, possibly due to the anti-clotting property and biomolecule-binding capabilities of heparin. These results shed light on the importance of biophysical and biochemical properties of nanofibers in the regulation of cell infiltration into 3-D scaffold and tissue remodeling, and emphasize that complex 3-D nanofibrous
Acknowledgement
This work was supported in part by a grant from NIH (HL083900, to S.L.), a grant from the Office of the Surgeon General of the Air Force (grant number FDG200712A, to J.S.) and an NIGMS-IMSD Training Grant (GM56847, to R.R.R.J.).
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